This invention relates to nucleic acid and amino acid sequences of a human E1-like protein and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative, immunological, neurological, and reproductive disorders.
The ubiquitin conjugation system (UCS) plays a major role in the selective degradation of cellular proteins in eukaryotes. UCS targets abnormal proteins for destruction and regulates the turn-over of proteins that control gene expression and cell cycle progression. For example, UCS mediates the degradation of mitotic cyclins, oncoproteins, tumor suppressors, viral proteins, transcription factors, and cell surface receptors. The timed destruction of these regulatory proteins by UCS is critical for ensuring normal cellular function. (Ciechanover, A. (1994) Cell 79:13-21; and Rolfe, M. et al. (1997) J. Mol. Med. 75:5-17.)
Ubiquitin (Ub) conjugation and protein degradation occur in several steps. (Jentsch, S. (1992) Annu. Rev. Genet. 26:179-207). First, Ub, a small, abundant protein, is activated by ubiquitin-activating enzyme (E1) in an ATP-dependent reaction. This reaction joins the C-terminus of Ub to the thiol group of a cysteine residue in E1. The product of this reaction is a high-energy E1-Ub thiolester intermediate. In a similar reaction, Ub is subsequently transferred from E1 to one of several Ub-conjugating enzymes (E2). Ub is then transferred from E2 in a reaction that joins the C-terminal glycine of Ub to an internal lysine of a protein targeted for destruction. In most cases, multiple ubiquitin moieties are transferred to the target protein. In some instances, additional factors called ubiquitin-ligases (E3) are required for target recognition. The ubiquitinated protein is then degraded by the proteasome, a large complex of up to 20 proteases and accessory factors. Following degradation of the target protein, Ub is released and reutilized.
Modifications of this pathway occur primarily in the steps involving E2 activity. A large number of structurally related, yet functionally distinct E2 enzymes have been identified across species. (Jentsch, supra.) These enzymes operate in distinct cellular compartments on specific target proteins, suggesting that E2 determines the selectivity of protein degradation.
E1 structure and function are conserved among various species including mammals, such as human and mouse; lower eukaryotes, such as yeast and nematode; and plants, such as wheat and wall cress. (Jentsch, supra; and Wilson, R. et al. (1994) Nature 368:32-38.) E1 is about 100 kilodaltons and contains an ATP-binding consensus sequence and an active site consensus sequence which includes the key cysteine residue to which Ub joins. No more than two different E1 proteins have been identified in a single species, suggesting that the general mechanism of Ub activation is common to all UCS pathways. However, in budding yeast, two E1 enzymes, Uba1p and Uba2p, have been identified, and both are essential for yeast viability, suggesting that these two proteins have distinct, nonoverlapping functions. (Dohmen, R. J. et al. (1995) J. Biol. Chem. 270:18099-18109.)
Although E2 appears to play the primary role in determining the activity of its cognate UCS, localization and modification of E1 may also influence this activity. For example, human E1 exists as two isoforms, E1a and E1b. E1a is localized primarily to the nucleus, while E1b is localized to the cytoplasm. E1a exists predominantly in a phosphorylated form, while E1b is mainly nonphosphorylated. The first 11 amino acids of E1 a contain a phosphoserine at residue 4 and a nuclear localization signal immediately following. Nuclear localization is required for phosphorylation, indicating that phosphorylation occurs within the nucleus itself. (Stephen, A. G. et al. (1997) J. Biol. Chem. 272:10895-10903.) Additional evidence suggests that the mitosis-specific kinase p34cdc2 may phosphorylate E1a in a cell cycle dependent manner. (Nagai, Y. et al. (1995) J. Cell Sci. 108:2145-2152.)
The diverse and complex roles of UCS have been elucidated using mutant mammalian cell lines that contain thermolabile E1. For example, the mouse cell line ts85 arrests in G2 phase of the cell cycle, suggesting that E1 activates Ub in a pathway required for cell cycle progression. (Jentsch, supra.) The CHO hamster cell line, ts20, is defective in the maturation of autophagic vacuoles which degrade proteins and subcellular organelles in response to stress conditions. (Lenk, S. E. et al. (1992) J. Cell Biol. 301-108.) In addition, mouse L-cells are defective in DNA replication, and this defect is rescued by either human or mouse E1 encoded by the A1S9 gene. Interestingly, A1S9 maps to a region of the mouse Y chromosome that is involved in spermatogenesis, and A1S9 is expressed in testis, suggesting that E1 encoded by A1S9 may play a specialized role in DNA replication events that occur during spermatogenesis. (Kay, G. F. et al (1991) Nature 354:486-489.)
UCS has been implicated in the molecular mechanisms and pathology of various diseases, and elements of UCS are therefore likely targets for therapeutic intervention. For example, agents that block UCS may allow the persistence of molecules that control cell proliferation. One such molecule is the tumor suppressor protein, p53, which is normally degraded by UCS. Inhibition of p53 degradation may prevent tumor cell proliferation. Likewise, inhibitors of mitotic cyclin degradation would also prevent cell cycle progression. (Rolfe et al. supra.) Another application for therapies that target UCS is in the treatment of immune disorders. For example, UCS mediates the proteolytic activation of NF-xcexaB, a key transcriptional regulator of genes involved in the immune response. Different therapeutic strategies that target this activation step could either stimulate the immune response or suppress autoimmune or inflammatory disorders. (Ciechanover, supra.) Finally, agents that block UCS may be valuable in the treatment of neurodegenerative disorders such as Alzheimer""s disease, in which abnormally high levels of ubiquitin and ubiquitin-conjugated proteins are observed in diseased tissue. (Muller, S. and Schwartz, L. M. (1995) Bioessays 17:677-684.) In fact, an E1-like protein, APP-BP1, has been found that specifically binds to xcex2-amyloid precursor protein. This protein is processed to form xcex2-amyloid, a major component of the plaque-like deposits that characterize Alzheimer""s brain pathology. (Chow, N. et al. (1996) J. Biol. Chem. 271:11339-11346.)
The discovery of a new human E1-like protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of cell proliferative, immunological, neurological, and reproductive disorders.
The invention is based on the discovery of a new human E1-like protein (HELPR), the polynucleotides encoding HELPR, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative, immunological, neurological, and reproductive disorders. The invention features a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist of the polypeptide.
The invention also provides a method for treating or preventing a cell proliferative disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing an immunological disorder associated with decreased expression or activity of HELPR, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing an immunological disorder associated with increased expression or activity of HELPR, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing a neurological disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing a reproductive disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for detecting a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.